The immune synapses reveal aberrant functions of CD8 T cells during chronic HIV infection

Chronic HIV infection causes persistent low-grade inflammation that induces premature aging of the immune system including senescence of memory and effector CD8 T cells. To uncover the reasons of gradually diminished potency of CD8 T cells from people living with HIV, here we expose the T cells to planar lipid bilayers containing ligands for T-cell receptor and a T-cell integrins and analyze the cellular morphology, dynamics of synaptic interface formation and patterns of the cellular degranulation. We find a large fraction of phenotypically naive T cells from chronically infected people are capable to form mature synapse with focused degranulation, a signature of a differentiated T cells. Further, differentiation of aberrant naive T cells may lead to the development of anomalous effector T cells undermining their capacity to control HIV and other pathogens that could be contained otherwise.


March 2021
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Life sciences Behavioural & social sciences Ecological, evolutionary & environmental sciences
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Life sciences study design
All studies must disclose on these points even when the disclosure is negative. Due to a limited cell availability, only a single sample was tested per individual. Rather than technical replicates performed with the cells from the same individual, samples from several individuals were tested. At least three independent donors were tested per experiment and donor group unless stated otherwise. All reported independent experiments are successful We have three observational study groups, namely, HIV+, HIV+ treated with antiretroviral therapy, and HIV uninfected donors. For each cohort, deidentified and coded samples were randomly chosen from available samples and allocated to the corresponding donor group The sample collection, deidentification and allocation to observational donor groups was performed by personnel that were not involved into the sample analysis.
anti Note that full information on the approval of the study protocol must also be provided in the manuscript.

Flow Cytometry
Plots Confirm that: The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).
All plots are contour plots with outliers or pseudocolor plots.
A numerical value for number of cells or percentage (with statistics) is provided.

Methodology
Sample preparation

Software
Cell population abundance

Gating strategy
The antibody has been previously validated in literature (Chen J.W. et al. The Journal of Cell Biology (1985) v. 101: 85-95) and in our previous work to study T cell degranulation using functional assay and glass-supported lipid bilayers (Betts M.R. et al. Journal of Immunological Methods (2003) v. 281: 65-78;Steblyanko M. et al. J Vis Exp. (2018) 137:58143). In current study, the antibody utilized for monitoring T cell degranulation on glass-supported lipid bilayers.
Hybridoma OKT3 producing antibodies recognizing human CD3zeta and hybridoma TS2/4.1.1 secreting antibodies against human LFA-1 were purchased from ATCC. Hybridoma H4A3 secreting antibody against CD107a (LAMP-1) was kindly provided by Dr. J. Thomas August, Department of Pharmacology and Molecular Sciences, Johns Hopkins Medical School. Hybridoma YN1.1 (ATCC) producing antibodies against ICAM-1 was kind gift from Michael Dustin, Skirball Institute of Biomolecular Medicine, New York University.
All antibody producing hybridoma cell lines were not authenticated. The antibody produced by the hybridoma cell lines were purified and validated.
The hybridoma cell lines were mycoplasma negative.
None of hybridoma cell lines used in the study are listed in the ICLAC register.
Samples obtained from deidentified HIV-and HIV+ individuals over the age of 18 were used for these studies. Relevant information adhering to BRISQ Tier 1 requirements provided in the manuscript.
All samples used in these studies were derived from IRB approved cohorts of patients at the University of Pennsylvania, and the University of Toronto. HIV+ and HIV-donors were recruited into these cohorts based on parameters established in their respective studies at each institution. No selection biases have been identified.
All enrolled participants provided written informed consent conform to Helsinki Declaration, and the protocol used was approved by the Institutional Review Board of the University of Pennsylvania (IRB 809316) and the University of Toronto (REB 12-378). HIV+ donors did not receive compensation beyond minimal costs associated with travel and time on the donation day. Healthy HIV-negative PBMC samples were obtained from the University of Pennsylvania Human Immunology Core. HIVdonors received $175 for apheresis donation to the University of Pennsylvania Center for AIDS Research Human Immunology Core. All data specimens were coded to protect confidentiality.
Cryopreserved PBMC were thawed, rested overnight, and sorted. Full details are provided in the Methods section of the manuscript. Analysis of LFA-1 expression: the analyzed CD8 T cell subsets are purified by magnetic sorting as described in the manufactiurer's protocol. The cells were live stained for 30 minutes at 4oC, washed, fixed, resuspended in FACS buffer and analyzed by flow cytometry.
Facs Aria, BDLSRFortressa, BD LSRII cytometers Becton Dickinson Diva software, FlowJo (v. 9.9.4) The recovery of the different populations varied based on the memory subset sorted. All sorted populations were 99% pure or higher.
Populations of naive T cells were sorted based on the gating strategy shown in Supplementary Information. Analysis of LFA-1